Approximately 1% of all human cancers are soft tissue sarcomas, which consists of more than 50 different tumor types. Soft tissue sarcomas are often malignant up to 50% of them recur or metastasize after the manifestation (Wibmer, C. 2010). Liposarcoma is the second common soft tissues sarcoma type, and depending on liposarcoma subtype, such as de-differentiated liposarcomas, it might present very aggressive disease course (Dalai, K. M. 2006). At the moment, surgical removal of tumor and radiotherapy are used to treat local disease. Chemotherapy is included in treatments in metastatic disease but responses are rarely achieved (D'Angelo, S. P. 2014).
Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract (GI) and it has been proposed to arise from progenitor cells of the interstitial cells of Cajal (Sircar, K. 1999). Strong expression of the KIT receptor tyrosine kinase (RTK) is typical for GISTs and about 80% of these tumors have a gain-of-function mutation in the KIT proto-oncogene (Hirota, S. 1998). Many GISTs lacking KIT mutation have activating mutations in the platelet-derived growth factor receptor α (PDGFRA) (Heinrich, M. C. 2003). PDGFRA is structurally related to KIT and both of these receptors belong to the same RTK family (Yarden, Y. 1987). Imatinib mesylate, a kinase inhibitor, has been known to significantly inhibit GISTs, presumably through inhibition of the KIT and PDGFRA (Heinrich, M. C. 2003). Other effective kinase inhibitors, used for the treatment of GIST, are multikinase inhibitors sunitinib and regorafenib that are used for the treatment of patients that imatinib has no effect or who have ceased to respond to imatinib (Demetri, G. D. 2006; Demetri, G. D. 2013). Some of GISTs do not respond for kinase inhibitors or they develop a resistance over time, so it is necessary to find new drug targets for GIST patients (Heinrich, M. C. 2006; Maleddu, A. 2009).
Immunohistochemical markers are crucial to the diagnostic of GIST. CD117 antibody which is targeted against an antigen in the KIT receptor is the most traditional diagnostic marker for GIST (Kindblom, L. G. 1998). High level of KIT expression is typical for GISTs which commonly harbor a KIT gene mutation in exon 9, 11, 13, or 17 (Heinrich, M. C. 2002). One of the most sensitive markers of GISTs is anoctamin 1 (ANO1; also known as DOG1) and it is considered to be even more specific marker of GIST than KIT (West, R. B., 2004; Espinosa, I. 2008; Liegl, B. 2009). Protein kinase C theta (PKCθ) is presented in most GISTs and is suggested as a useful GIST marker (Duensing, A. 2004). PKCθspecificity for GIST is however relatively low (Novelli, M. 2010). Another marker used to evaluate GISTs is the hematopoietic progenitor cell antigen CD34, whereas smooth muscle antigens, such as smooth muscle actin, desmin, embryonic myosin and heavy caldesmon are not often expressed in GISTs (Miettinen, M. 1995; Miettinen, M. 2006).
Phosphodiesterases (PDEs) selectively catalyse the hydrolysis of the 3′-phosphoester bond of the 3′,5′-purine ribose cyclic monophosphates cAMP and cGMP. PDEs regulate the cellular levels, localisation and signalling of cAMP and/or cGMP by controlling their degradation (Beavo, J. A. 1995). There is totally 11 different PDE families and many of these families have multiple transcription variants (Conti, M. 2007). PDE3 family includes two proteins, PDE3A and PDE3B, which show structural similarity (Degerman, E. 1997). PDE3A is abundant in cardiac myocytes, vascular smooth muscle cells and platelets (Shakur, Y. 2001). PDE3 inhibitors have been reported to enhance myocardial contractility and induce vascular and airway smooth-muscle relaxation (Halpin, D. M. 2008). Different PDE3 inhibitors have been used to treat heart failure and intermittent claudication (Dawson, D. L. 1998; Liu, Y. 2001). PDE3B is expressed in pancreatic beta-cells, hepatocytes and adipocytes, in which it regulates metabolic processes such as insulin secretion of beta-cells, glycogenolysis of liver cells and lipolysis in adipocytes (Resjö, S. 1999). In obese diabetic mice, PDE3 inhibitor cilotazol has been showed to improve glucose intolerance and insulin resistance by suppressing macrophage secreted TNFα-induced inflammation in adipose tissue (Wada, T. 2013).
PDE3A is phosphorylated and activated by AKT in mammalian oocyte maturation. (Han, S. J. 2006). Similarly, PDE3B is showed to be phosphorylated and activated by AKT after insulin stimulation in adipocytes (Degerman, E. 1998). The phosphatidyl-inositol-3-kinase (PI3-K)/AKT pathway is also activated by mutated KIT and PDGFRA receptors in GISTs (Duensing, A. 2004). In platelets PKC is involved in regulation of PDE3A activity (Hunter, R. W. 2009). PDE3A has been shown to be expressed in gastric smooth muscle, where PDE3A is phosphorylated by protein kinase A (Murthy, K. S. 2002). In addition, PDE3A showed strong expression in the gastric antrum of a murine model of GIST (KITK641E mouse), and was associated with the gene expression profile of interstitial cells of Cajal (Gromova, P. 2009).
Anagrelide hydrochloride is a molecule which targets PDE3 enzymes and is able to lower the amounts of platelets. It has been used in patients with myeloproliferative disorders and especially for the treatment of essential thrombocytosis (Fruchtman, S. M. 2005; Harrison, C. N. 2005). Anagrelide hydrochloride inhibits the cyclic AMP phosphodiesterase activity in platelets, which further elevates the cAMP levels in these cells (Gillespie, E. 1988; Seiler, S. 1987).
In the present invention, we'll demonstrate that PDE3A and PDE3B are specific markers for GIST. PDE3 proteins are also expressed often in liposarcomas and less frequently in other cancer types. Inhibition of PDE3 activity with anagrelide hydrochloride halts cell proliferation in GIST cell lines and significantly decreases cell proliferation in liposarcomas cell line. When anagrelide hydrochloride, a PDE3 specific inhibitor, was used in combination with imatinib mesylate in GIST cell lines, synergistic effect could be seen. The GIST cells were more apoptotic and cell proliferation decreased more than using either of the drugs alone. Our findings here show that PDE3A and PDE3B can be considered new immunomarkers and drug targets, and PDE3 inhibitor anagrelide hydrochloride has strong therapeutic potential for the treatment of PDE3 expressing solid tumors.